Transcriptomic Analysis of Adipose-Derived Stem Cell Therapy Modulating B Cell Immune Tolerance in Vascularized Composite Allotransplantation.

Abstract

Adipose-derived stem cells (ADSCs) have been shown to prolong vascularized composite allotransplantation (VCA) survival in rodent hindlimb transplantation. However, the mechanisms by which ADSCs modulate B cell responses and induce immune tolerance remain unclear. This study used next-generation sequencing (NGS) to investigate B cell gene expression after ADSC treatment. A rodent orthotopic hind-limb transplantation model was established using Brown-Norway to Lewis rats. The tolerance treatment group received a combination of ADSCs, short-term cyclosporine A (CsA) and anti-lymphocyte serum (ALS), while the control rejection group received no treatment. B cells were isolated from the spleens of rejection control group at 10-14 days post-transplantation when rejection was diagnosed, or tolerance group at least ≥100 days post-transplantation without rejection. RNA sequencing (RNA-Seq) was conducted to assess transcriptomic differences. Differentially expressed genes (DEGs) were analyzed using the EBSeq bioinformatics tool and pathway enrichment analyses, with validation by qPCR. RNA-Seq identified 94 DEGs (out of 13,284 mRNAs) between the tolerance and rejection groups. Gene ontology and KEGG pathway analyses revealed significant enrichment in pathways associated with cytokine secretion, B cell receptor signaling, and humoral immunity. The tolerance group exhibited upregulation of key chemokines (e.g., Cxcl1, Cxcl2, Ccl4) and downregulation of pro-inflammatory genes (e.g., Tlr3, Siglec5, C3). qPCR validation confirmed the RNA-Seq findings. These results suggest that ADSCs modulate B cell-mediated immune responses and promote a shift toward an anti-inflammatory, tolerogenic profile. ADSC-based therapy combined with short-term immunosuppression promotes immune tolerance in VCA by modulating B cell-related gene expression. These findings suggest that targeting B cell-mediated pathways may provide a novel approach may offer a novel therapeutic strategy for clinical application in VCA.